IL-10 producing type 1 regulatory T cells (Tr1 cells) play an important role in immune regulation; thus, Tr1 cell induction and expansion are potential approaches to treat immune-mediated disorders. However, the mechanisms controlling Tr1 cell differentiation and function are largely unknown. During our previous funding cycle we found that the transcription factor AHR controls Tr1 cell differentiation. In addition, we recently found that IL-27 and AHR control the expression of the ectonucleotidase CD39 in Tr1 cells and dendritic cells (DCs). CD39 degrades pro-inflammatory extracellular adenosine triphosphate (eATP) into adenosine monophosphate (AMP); AMP is then used by the ecto-5'-nucleotidase CD73 to synthesize immunosuppressive adenosine. Our preliminary data suggest that hypoxia and eATP (both of which are detected in inflamed tissues) suppress Tr1 cell differentiation in a HIF1?-dependent manner. Moreover, preliminary experiments suggest that CD39 contributes to the suppressive activity of Tr1 cells independently of IL-10, through the production of adenosine in cooperation with CD73 expressed by responder T cells (Tresp) and DCs. Based on these observations, we hypothesize that the balance between AHR/CD39 and eATP/HIF1? activation controls Tr1 cell differentiation, stability and function. Our specific aims are: Specific Aim 1: Hw does AHR/CD39 and eATP/HIF1? activation control Tr1 cell differentiation? Our preliminary data suggest that eATP and hypoxia (metabolic features of inflamed tissues) suppress Tr1 cell differentiation by inhibiting AHR through a mechanism mediated by the transcription factor HIF1?. Thus, we propose to: 1) Examine the role of CD39 on limiting the inhibitory effects of eATP on Tr1 cell differentiation. 2) Analyze the effects of eATP and hypoxia on AHR signaling and Tr1 cell differentiation, and the role of HIF1? in these effects. 3) Study the effects of eATP and hypoxia on the transcriptional program of Tr1 cells. Specific Aim 2: How does CD39 expressed by Tr1 cells contribute to their suppressive function? Our preliminary data suggest that CD39 participates in the suppressive function of Tr1 cells. Thus, we propose to: 1) Study the function of CD39 on Tr1 suppressive activity. 2) Analyze the effects of eATP depletion on Tr1 cell stability and function. 3) Examine the contribution of adenosine generated by CD73 in Tresp in cooperation with CD39 expressed by Tr1 cells on their suppressive function. Specific Aim 3: What is the role of DCs on Tr1 cell differentiation and function? IL-27 signaling in DCs limits inflammation in a CD39-dependent manner. Our preliminary data suggest that CD39 and CD73 in DCs control Tr1 cell differentiation and function. Thus, we propose to: 1) Study the role of CD39 expressed by DCs on Tr1 cell generation and stability. 2) Analyze the role of CD39 and CD73 in DCs on Tr1 suppressive function. In summary, this project studies positive (AHR/CD39) and negative (eATP/HIF1?) regulators of Tr1 cell differentiation, their physiological roles and their potential value for the therapeutic modulation of Tr1 cells.